Custom-formulated phospholipid microbubbles and methods and uses thereof

ABSTRACT

A phospholipid microbubble comprising a shell which comprises a plurality of polyunsaturated fatty acid (“PUFA”)-containing phospholipids, and a core of paramagnetic gas surrounded by the shell comprising the plurality of PUFA-containing phospholipids. The present invention also provides methods of delivering a prophylactically or therapeutically effective amount of PUFA to an area of disease or injury in a subject. The present invention also provides methods of preventing or treating a disease in a subject using a prophylactically or therapeutically effective amount of the aforementioned phospholipid microbubbles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Utility patent application Ser.No. 13/010,601 which application claims benefit under 35 U.S.C. §119(e)to U.S. Provisional Patent Application No. 61/296,767, filed Jan. 20,2010, which is incorporated by reference in its entirety herein.

Throughout this application, several patents, patent applications andreferences are referenced herein. Disclosures of these patents, patentapplications and references in their entirety are hereby incorporated byreference into this application.

BACKGROUND OF THE INVENTION

The present invention generally relates to new pharmacologicalformulations of phospholipid microbubbles comprising a plurality ofphospholipids that allow for the rapid, efficient and target delivery ofpolyunsaturated fatty acids (“PUFA”) to areas of disease or injury in asubject for diagnostic, prophylactic and/or therapeutic aims.

Omega-3 PUFA:

Omega-3 PUFA is an essential nutrient critical for the maintenance ofhuman health. Interest in omega-3 PUFA stems from the first observationsby Bang and Dyerberg during the 1970s that death from coronary arterydisease (CAD) is extremely rare among the Greenland Eskimos who consumea diet rich in EPA and DHA. Since that seminal discovery, intense basicand animal research has revealed a multitude of beneficial effects ofomega-3 PUFA. These positive effects include anti-inflammatory,anti-cancerous, immunomodulatory, anti-diabetic, anti-thrombotic andanti-arrhythmic properties, to name but a few. In addition, numerousepidemiological studies and randomized controlled trials have beenconducted that demonstrated similar wide-ranging health benefits inhumans. For example, the landmark GISSI-P (Gruppo Italiano per lo Studiodella Streptochinasi nell'Infarto Miocardico—Prevenzione) preventionstudy randomized 11,324 patients with pre-existing CAD to 850 mg ofomega-3 PUFA resulted in a 45% reduction in sudden cardiac death and 30%reduction in cardiovascular death. (Dietary supplementation with n-3polyunsaturated fatty acids and vitamin E after myocardial infarction:results of the GISSI-Prevenzione trial. The Lancet, 1999; 354:447-55).Moreover, the benefits of these essential nutrients appear not to belimited solely to the cardiovascular system but extend to virtuallyevery organ system in the human body. (Simopoulos A P. Omega-3 fattyacids in inflammation and autoimmune diseases. J Am Coll Nutr 2002;21:495-505).

Tragically, the dietary practices of modern industrial society over thelast century have been moving towards a diet that is severely deficientin omega-3 PUFA. This nutritional deficit is further compounded by therise in the consumption of “processed” foods containing hydrogenatedtrans-fatty acids and omega-6 PUFA that possess antagonistic propertiesto omega-3 PUFA. Moreover, the content of omega-3 PUFA in meat productswas decreased by the modern practice of raising animals fed withcorn-base stock in feedlots rather than grass fed on the open field.Together, these changes resulted in a striking imbalance ofpro-inflammatory and anti-inflammatory types of PUFA in the typicalmodern diet. Up until only recently, humans evolved and flourished on adiet with a ratio of omega-6:omega-3 fatty acids close to 1:1. It hasbeen estimated that today this ratio has risen dramatically to as highas 20:1. (Simopoulos A P. The importance of the ratio of omega-6/omega-3essential fatty acids. Biomed Pharmacother 2002; 56:365-79). As aconsequence of these changes in eating habits, food processing and inagriculture, entire populations are now at risk of developing a widespectrum of inflammatory, autoimmune and degenerative diseases.

Despite their wide ranging health benefits, the therapeutic use ofomega-3 PUFA is currently limited by the lack of a rapid and effectivemeans of delivery. Oral supplementation of omega-3 PUFA requires weeksto months to attain adequate blood levels and to be incorporated intocellular membranes to exert their effects. Furthermore, the “fishy”unpleasant taste of omega-3 PUFA makes it difficult for patients tomaintain strict compliance. More importantly, the delayed onset ofaction of oral supplementation has precluded their use in the treatmentof acute emergent medical conditions such as acute myocardial infarctionand cerebral vascular accidents and their complications. To achieve amore rapid onset of action, intravenous infusion of omega-3 PUFA hasbeen used successfully to terminate and prevent life-threateningventricular arrhythmias that occurred in the setting of AMI in variousanimal models and in humans. (McGuinness J, Neilan T G, Sharkasi A,Bouchier-Hayes D, Redmond J M. Myocardial protection using an omega-3fatty acid infusion: Quantification and mechanism of action. J ThoracCardiovasc Surg 2006; 132:72-9; Billman G E, Hallaq H, Leaf A.Prevention of Ischemia-Induced Ventricular Fibrillation by {omega}3Fatty Acids. PNAS 1994; 91:4427-30; and Schrepf R, Limmert T, ClausWeber P, Theisen K, Sellmayer A. Immediate effects of n-3 fatty acidinfusion on the induction of sustained ventricular tachycardia. Lancet2004; 363:1441-2). Omega-3 PUFA have also been shown to be exertimmuno-modulatory activity in preventing heart transplant rejection inrats. (Grimminger F, Grimm H, Fuhrer D, et al. {omega}-3 Lipid Infusionin a Heart Allotransplant Model: Shift in Fatty Acid and Lipid MediatorProfiles and Prolongation of Transplant Survival. Circulation 1996;93:365-71). However, omega-3 PUFA administered by the intravenous routehave to be in the form of triglycerides rather than in their freebioactive non-esterified forms. Enzymatic hydrolysis of thesetriglycerides by endogenous lipases is needed for their release andactivation. Because of interindividual differences in the rate of theirmetabolism, the blood levels of omega-3 PUFA after intravenous infusionwere found to be highly variable. (Schrepf R, Limmert T, Claus Weber P,Theisen K, Sellmayer A. Immediate effects of n-3 fatty acid infusion onthe induction of sustained ventricular tachycardia. Lancet 2004;363:1441-2). Because of the slow and variable rate of hydrolysis and theneed to saturate the entire body, a prolonged infusion that lasts anexcess of 90 minutes is generally required to ensure that adequatetherapeutic levels can be achieved. This unpredictable bioavailabilityprevents their use in emergent clinical situations such as heartattacks, strokes and their arrhythmic sequela.

Ultrasound Microbubbles:

Prior art ultrasound microbubbles are gas-filled vesicles havingdiameters on the order of less than 10 microns enclosed in abiocompatible shell composed of a lipid, protein or polymer. The gascore is made of an inert high-molecular weight gas (i.e.,perfluorocarbons, sulfur hexafluoride) such as to typically minimizevolume loss and to ensure stability. The small size of the microbubblesallow for their unimpeded passage through the microcirculation of thelungs to any organs of the body by intravenous administration. Variousformulations of these microbubbles are currently employed as contrastagents to enhance diagnostic images obtained by ultrasonography, as inechocardiography to help visualize the left ventricular cavity of theheart and to assess myocardial perfusion.

More recently, these microbubbles have been modified for therapeutic useas vehicles for drug delivery and for gene therapy. (Feinstein S B. Thepowerful microbubble: from bench to bedside, from intravascularindicator to therapeutic delivery system, and beyond. Am J Physiol HeartCirc Physiol 2004; 287:H450-7). The dual versatility of microbubbles formolecular imaging and target drug delivery termed “theranostic”applications is only now beginning to be exploited. (Pan D, Lanza G M,Wickline S A, Caruthers S D. Nanomedicine: Perspective and promises withligand-directed molecular imaging. European Journal of Radiology 2009;70:274-85). By focusing the ultrasound energy at a desired target site,higher local concentrations of a therapeutic agent may be achieved. Forexample, U.S. Pat. No. 5,558,092 describes compositions, methods andapparatus for carrying out diagnostic and therapeutic ultrasound.Contrast materials loaded with a therapeutic agent are imaged usingdiagnostic ultrasound waves, and once seen accumulating in a desiredarea, are ruptured using ultrasonic waves to generate enhancedcavitation or the targeted release of an agent into the region. Couplingdiagnostic and therapeutic ultrasound modes provides additionaladvantages of monitoring efficacy and dose adjustments. However, themajor limiting aspect of the current art remains poor efficiency ofdelivery.

SUMMARY OF THE INVENTION

In consideration of the above problems, in an aspect of the presentinvention, a phospholipid microbubble comprising a shell which comprisesa plurality of polyunsaturated fatty acid (“PUFA”)-containingphospholipids, and a core of paramagnetic gas surrounded by the shellcomprising the plurality of PUFA-containing phospholipids.

Each of the phospholipids within the shell comprises a polar head group,and two non-polar fatty acid tails, the two non-polar fatty acid tailsare linked to the polar head group via a glycerol linkage. Thephospholipids can be customized to identify and/or treat a specificdisease or injury by selecting a type of polar head groups and/ornon-polar fatty acid tails. By way of example, the phospholipidmicrobubbles can be made of PS polar head groups linked to non-polarfatty acid tails of omega-3 PUFAs (such as EPA, DHA, or a combinationthereof) to treat acute myocardial infarction and prevent its arrhythmiccomplications. Examples of polar head groups that can be used tocustomize the phospholipids are PS, PC, PE, PI, or a combinationthereof, in varying proportions depending on the specific diagnostic,prophylactic and/or therapeutic aims. Examples of non-polar fatty acidtails that can be used to customize the phospholipids are omega-3 PUFA(e.g., EPA, DHA), omega-3 PUFA precursors (e.g., α-linolenic acids),omega-3 PUFA-derived metabolites (e.g., resolvins, neuroprotectins,lipoxins, neuroprostanes), omega-6 PUFA, omega-9 PUFA, conjugatedlinoleic acids, conjugated linoleic acid isomers, or a combinationthereof, in varying proportions depending on the specific diagnostic,prophylactic and/or therapeutic aims.

In another aspect, the plurality of PUFA-containing phospholipidscomprises a plurality of polar head groups, said plurality of polar headgroups comprising phosphatidylserine (“PS”), phosphadtidylcholine(“PC”), phosphatidyl-ethanolamine (“PE”), phosphatidylinositol (“PI”),or a combination thereof.

In another aspect, the plurality of PUFA-containing phospholipidscomprises a plurality of non-polar fatty acid tails, the plurality ofnon-polar fatty acid tails comprising an omega-3 PUFA, an omega-3 PUFAprecursor, an omega-3 PUFA-derived metabolite, an omega-6 PUFA, anomega-9 PUFA, a conjugated linoleic acid, a conjugated linoleic acidisomer, or a combination thereof.

In another aspect, the omega-3 PUFA is selected from eicosapentaenoicacid (“EPA”), docosahexaenoic acid (“DHA”), or a combination thereof.

In another aspect, the omega-3 PUFA precursor is an □-linolenic acid.

In another aspect, the omega-3 PUFA-derived metabolite is selected froma resolvin, a neuroprotectin, a lipoxin, a neuroprostane, or acombination thereof.

In another aspect, the paramagnetic gas is selected from aperfluorocarbon compound, xenon, hyperpolarized xenon, or other suitablegas that enhances target delivery and/or permits detection andmonitoring by MRI.

In another aspect, the phospholipid microbubble in a micellar form or aliposomal form.

In another aspect, the plurality of PUFA-containing phospholipidscomprises at least one PS-DHA phospholipid, PS-EPA phospholipid, PC-DHAphospholipid, or PC-EPA phospholipid.

In another aspect, the phospholipid microbubble further comprises adrug, a fat-soluble compound, an antioxidant, an antibody or a fragmentthereof, or a specific ligand is conjugated to the phospholipidmicrobubble. For example, vitamin A, vitamin D, vitamin E, vitamin K,resveratrol, astaxanthin, or a combination thereof can be conjugated tothe phospholipid microbubbles to achieve synergistic or additiveeffects. With respect to the specific ligand, such specific ligand canbind to an antibody, such as a monoclonal or polyclonal antibody.

In another aspect of the present invention, a method of delivering aprophylactically or therapeutically effective amount of PUFA to an areaof disease or injury in a subject, comprising the steps of administeringto the subject a plurality of phospholipid microbubbles which comprisesa shell comprising a plurality of PUFA-containing phospholipids, and acore of paramagnetic gas surrounded by the shell comprising saidplurality of PUFA-containing phospholipids; allowing the phospholipidmicrobubbles to reach the area of disease or injury; and applyingultrasound to the area of disease or injury to explode the phospholipidmicrobubbles.

In another aspect, the area of disease or injury is the heart.

In another aspect, the plurality of phospholipid microbubbles comprisesa micellar form of the phospholipid microbubbles, a liposomal form ofphospholipid microbubbles, or a combination thereof.

In another aspect of the present invention, a method of preventing ortreating a disease in a subject, comprising the steps of administeringto the subject a prophylatically or therapeutic effective amount of aplurality of phospholipid microbubbles which comprises a shellcomprising a plurality of PUFA-containing phospholipids, and a core ofparamagnetic gas surrounded by said shell comprising the plurality ofPUFA-containing phospholipids; and applying ultrasound to thephospholipid microbubbles to explode the phospholipid microbubbles inthe subject.

In another aspect, the disease can be an inflammatory, autoimmune,neoplastic or degenerative disease. Further, the disease can be lupuserythematosis, multiple sclerosis, rheumatoid arthritis, Crohn'sdisease, ulcerative colitis, psoriasis, diabetes mellitus, prostatecancer, breast cancer, depression, Alzheimer's disease or myocardialinfarction.

In another aspect, the administration is a single intravenousadministration of said plurality of phospholipid microbubbles.

In another aspect, the plurality of PUFA-containing phospholipidscomprising at least one PS-DHA phospholipid, PS-EPA phospholipid, PC-DHAphospholipid, or PC-EPA phospholipid.

In another aspect, the plurality of phospholipid microbubbles comprisesa micellar form of said phospholipid microbubbles, a liposomal form ofsaid phospholipid microbubbles, or a combination thereof.

In another aspect, the administration is a co-administration of amicellar form of said phospholipid microbubbles and a liposomal form ofsaid phospholipid microbubbles.

In another aspect, the co-administration is simultaneous, concurrent orsequential administration of said micellar form of said phospholipidmicrobubbles and said liposomal form of said phospholipid microbubbles.

A small therapeutically effective amount of these customizedphospholipid microbubbles is need to be administered intravenously tothe subject to achieve diagnostic, prophylactic and/or therapeuticeffects since they can be targeted to areas of disease or injury byusing focused ultrasound. Moreover, due to the paramagnetic nature ofthe microbubble core, the efficacy of prophylactic and/or therapeutictreatment can be further enhanced and monitored by magnetic resonanceimaging (“MRI”). This novel formulation strategy promises not only to bedisease-specific but also adaptable to treating a wide spectrum ofpathological conditions including inflammatory, autoimmune, neoplasticand degenerative diseases.

DEFINITIONS

The term “therapeutically effective amount” as used herein refers tothat amount sufficient to treat, manage or ameliorate a disease,disorder or injury in a subject.

The term “prophylactically effective amount” as used herein refers tothat amount sufficient to prevent a disease, disorder, or injury in asubject.

The term “subject” as used herein refers to an animal (e.g., a bird,which includes, but not limited to, a chicken, quail or turkey, or amammal), preferably a mammal which includes, but not limited to, anon-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, goat,rat, cat, dog, and mouse) or a primate (e.g., a monkey, chimpanzee, andhuman), and more preferably a human. In a preferred embodiment, thesubject is a human.

As used herein, the term “antibody” includes, but is not limited to,polyclonal antibodies, monoclonal antibodies, humanized or chimericantibodies and biologically functional antibody fragments sufficient forbinding of the antibody fragment to a protein. See, Harlow & Lane,Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988). A “specific ligand” for an antibody isthe composition of matter, for example in the blood of a subject, towhich an antibody binds with high affinity. Many descriptions of theterm “specific ligand” are available to those of skill in the art. See,e.g., van Oss C. J., “Nature of specific ligand-receptor bonds, inparticular the antigen-antibody bond.” J. Immunoassay 21(2-3):109-42(May-August 2000).

BRIEF DESCRIPTION OF THE FIGURES

For the purpose of illustrating the present invention, the drawingsreflect a form which is presently preferred; it being understood,however, that the invention is not limited to the precise form shown inthe drawings in which:

FIG. 1 below shows two exemplary molecular forms of the presentinvention comprising PUFA-containing phospholipids: (a) a micellar formcomprising a monolayer of phospholipids, and (b) a liposomal formcomprising a bilayer of phospholipids.

FIG. 2 below shows anti-arrhythmic effects of omega-3 PUFA-containingmicrobubbles in a dog with myocardial infarction. Specifically, threerepresentative leads (I, II and V) from a surface electrocardiographicrecording of a dog with myocardial infarction demonstrating conversionfrom ventricular tachycardia to normal sinus rhythm. Within a fewseconds after microbubbles administration and application oftransthoracic ultrasound over the region of infarct, abnormalventricular beats (V) were replaced by normal sinus beats (N). Fusionbeats (F) formed from a combination of V and N beats were observedduring the early part of the transition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention overcomes many of the existing limitations ofcurrent modes of delivery of PUFA (such as omega-3 PUFA) by preferablyincorporating them into phospholipid microbubbles and using focusedultrasound to target them to areas of disease or injury.

One of the goals of the present invention is to deliver PUFA (e.g.,omega-3 PUFA) as integral components of microbubbles to diseased orinjured areas for diagnostic, prophylactic and/or therapeutic purposes.This is achieved by fabricating microbubbles such that they can bedelivered efficiently by focused ultrasound and monitored by MRI. Thepreferred basic structure of these microbubbles is that of a shell whichcomprises a plurality of phospholipids, and a core of paramagnetic gassurrounded by the shell. Each phospholipid within the shell comprises aglycerol backbone to which a polar head group and two non-polar fattyacid tails are linked. The PUFA to be delivered can be linked to themiddle (sn-2) position of the glycerol backbone. The polar head group isin the first (sn-1) position while the third (sn-3) position can beeither a saturated or non-saturated fatty acid. The types of polar headand non-polar tails can be customized based on an understanding of theunderlying pathophysiology of the specific disease or injury to beprevented or treated. Alternatively, the PUFA-containing phospholipidscan be derived from a natural source of PUFA, such as those extractedfrom krill oil.

Prior art gas-encapsulated microbubbles with shells composed either ofalbumin or lipid have been shown to bind specifically to a variety ofinflammatory cells such as neutrophils, monocytes and macrophages and todamaged endothelial cells in vivo. (Lindner J R, Dayton P A, Coggins MP, et al. Noninvasive Imaging of Inflammation by Ultrasound Detection ofPhagocytosed Microbubbles. Circulation 2000; 102:531-8; Villanueva Md FF S, Jankowski Ms R J, Manaugh Bs C, Wagner PhD W R. Albumin MicrobubbleAdherence to Human Coronary Endothelium: Implications for Assessment ofEndothelial Function Using Myocardial Contrast Echocardiography. Journalof the American College of Cardiology 1997; 30:689-93; and Yanagisawa K,Moriyasu F, Miyahara T, Yuki M, Iijima H. Phagocytosis of ultrasoundcontrast agent microbubbles by Kupffer cells. Ultrasound in Medicine &Biology 2007; 33:318-25). Furthermore, these microbubbles are taken upor phagocytosed into the interior of these cells where they remainacoustically active. Based on these experimental observations, Lindnerand his group employed these microbubbles as ultrasound imaging agentsto detect areas of inflammation and injury. (Lindner J R, Song J, Xu F,et al. Noninvasive Ultrasound Imaging of Inflammation Using MicrobubblesTargeted to Activated Leukocytes. Circulation 2000; 102:2745-50). Themicrobubbles employed by this group are composed of a mixture ofphospholipids containing PC and PS polar head groups linked to stearateacids. Stearate acids are saturated fatty acids that are known topossess proinflammatory properties when they are cleaved and releasedfrom their parent phospholipid molecule by cellular enzymes calledphospholipases. This fact may account for the success of this imagingtechnique in detecting inflammation since the stearate acids withinthese microbubbles actually serve to amplify the inflammatory process.Unfortunately, an unanticipated consequence of these microbubbles isthat it tends to exacerbate the underlying inflammatory process that isbeing imaged, and, thus, explain why this technique has not been adoptedclinically for use in humans.

The phospholipid microbubbles of the present invention exploits theknown anti-inflammatory effects of omega-3 PUFAs and the proresolvingproperties of their metabolites, such as resolvins, neuroprotectins andlipoxins. (Serhan C N, Chiang N, Van Dyke T E. Resolving inflammation:dual anti-inflammatory and pro-resolution lipid mediators. Nat RevImmunol 2008; 8:349-61). Another potential mechanism of benefit relatesto the newly discovered anti-inflammatory metabolites formed fromperoxidation of omega-3 called neuroprostanes. (Musiek E S, Brooks J D,Joo M, et al. Electrophilic cyclopentenone neuroprostanes areanti-inflammatory mediators formed from the peroxidation of the omega-3polyunsaturated fatty acid docosahexaenoic acid. J Biol Chem 2008;283:19927-35). By altering a critical fatty acid component of the lipidshell, these novel formulations can be devoid of the adverse effects ofmicrobubbles made of proinflammatory saturated fatty acids. (Main M L,Goldman J H, Grayburn P A. Ultrasound contrast agents: balancing safetyversus efficacy. Expert Opinion on Drug Safety 2009; 8:49-56). Moreover,these new formulations can be used not only for image contrastenhancement but also for treatment purposes. In addition to usingfocused ultrasound delivery, the target specificity of thesemicrobubbles can be further enhanced by choosing the type of polar headgroups appropriate to the disease or injury being prevented or treated.For example, PS is a phospholipid which is an almost universalrecognition ligand for phagocytes mediating apoptosis, a cellularmechanism of program cell death that leads to the non-phlogistic healingof inflamed or injured tissues. (Gardai S J, Bratton D L, Ogden C A,Henson P M. Recognition ligands on apoptotic cells: a perspective. JLeukoc Biol 2006; 79:896-903). Thus, the incorporation of PS as polarhead groups in the microbubbles' phospholipid shell can enhancemicrobubble uptake by phagocytic cells within diseased or injured areas.In addition, the phospholipids released from these microbubbles can alsocompete with endogenous pro-inflammatory fatty acids (e.g., arachidonicacid) released during the inflammatory process, further promoting itsresolution.

Although microbubbles have been used for therapeutic applications suchas the delivery of genes, proteins and drugs, they have not been used inthe delivery of PUFA as described by the present invention herein.(Bekeredjian R, Grayburn P A, Shohet R V. Use of ultrasound contrastagents for gene or drug delivery in cardiovascular medicine. J Am CollCardiol 2005; 45:329-35, and Liu Y, Miyoshi H, Nakamura M. Encapsulatedultrasound microbubbles: Therapeutic application in drug/gene delivery.Journal of Controlled Release 2006; 114:89-99). All existing lipid-basedmicrobubbles in clinical use are composed of saturated fatty acids thatmay have adverse pro-inflammatory potential. This unrecognized sideeffect might have accounted for the slight increase in cardiovascularevents that prompted their current black box label. (Main M L, Goldman JH, Grayburn P A. Ultrasound contrast agents: balancing safety versusefficacy. Expert Opinion on Drug Safety 2009; 8:49-56). In addition, allexisting applications require either the intracellular or intranuclearpenetration of the contents of the microbubbles that severely limit theefficiency of delivery. In contrast, the present invention allows thetarget delivery of fatty acids to the surface of the cell membraneswithout the need to penetrate into the cytoplasm or nucleus, as it hasbeen shown that omega-3 PUFA exert their acute effects by partitioninginto the cell membranes and altering the physiochemical properties ofcaveolae and of ion-channels within the membrane lipid bilayer.(Weylandt K H, Kang J X, Leaf A. Polyunsaturated fatty acids exertantiarrhythmic actions as free acids rather than in phospholipids.Lipids 1996; 31:977-82; Pound E M, Kang J X, Leaf A. Partitioning ofpolyunsaturated fatty acids, which prevent cardiac arrhythmias, intophospholipid cell membranes. J Lipid Res 2001; 42:346-51; and LionettiV, Fittipaldi A, Agostini S, Giacca M, Recchia F A, Picano E. EnhancedCaveolae-Mediated Endocytosis by Diagnostic Ultrasound In Vitro.Ultrasound in Medicine & Biology 2009; 35:136-43). Therefore, thepresent invention is superior to previous methods in terms of rapidityand efficiency of delivery. The therapeutic effect is expected to occurwithin seconds and the total duration of administration is anticipatedto be not more than one minute rather than more than one hour bycurrently available methods. The targeted nature of delivery also limitsany potential systemic side effects related to treatments using thePUFA-containing microbubbles of the present invention. Moreover, becauseomega-3 PUFA are natural nutrients, they are devoid of immunogenicpotential and can thus be used in repeated administrations. Also, theuse of ultrasound for focused delivery of the present invention may notbe required since the polar head groups of the present invention can becustomized to specifically target areas of inflammation or injury.

The PUFA-containing microbubbles of the present invention can bepreferably formulated for intravenous administration as either a bolusor a continuous infusion, and preferably targeted to a specific organusing focused ultrasound. The composition of these microbubbles iscustomized for a specific disease or injury condition by formulating thetype of polar head group and non-polar fatty acid tails of theconstituent phospholipids. In addition, other fat-soluble compounds(e.g., antioxidants), the particular type of core gas (e.g., aperfluorocarbon compound, xenon, hyperpolarized xenon, or other suitablegas that enhances target delivery and/or permits detection andmonitoring by MRI) can be chosen to further enhance the diagnostic,prophylactic and/or therapeutic effect.

The PUFA-containing microbubbles can be administered in a micellar form,a liposomal form, or a combination thereof, in varying proportionsdepending on the specific diagnostic, prophylactic and/or therapeuticaims. The co-administration of the micellar and liposomal forms of thePUFA-containing microbubbles includes simultaneous, concurrent, orsequentially administration of the micellar and liposomal forms to thesubject. Simultaneous administration means administration of themicellar and liposomal forms in a single dosage form; concurrentadministration means administration of the micellar and liposomal formsat about the same time but in separate dosage forms; and sequentialadministration means administration of one of the forms, after which theother is administered. Sequential administration can also take the formof simultaneous or concurrent administration(s) of the micellar andliposomal forms, followed by cessation of the simultaneous or concurrentadministration(s) and then continued administration of one of the formsalone, or vice versa.

Methods that are well-known to the art can be used to fabricate thesemicrobubbles. For example, a variation of the method as described byHuang et al. can be used. (Huang S-L, McPherson D D, MacDonald R C. AMethod to Co-Encapsulate Gas and Drugs in Liposomes forUltrasound-Controlled Drug Delivery. Ultrasound in Medicine & Biology2008; 34:1272-80). Briefly, liposomes (or micelles) of the desiredcomposition are prepared from chloroform solutions by combining theappropriate molar amounts of the individual component phospholipids.Alternatively, a natural lipid mixture with the desired phospholipidcomposition can be obtained by extraction and distillation from naturalsources and be used as the starting material. After mixing in a glassvial, the organic solvent is removed by evaporation under argon gas in a50° C. water bath with constant rotation until a thin film of lipids isformed on the sidewall of the vial. The lipid film is then placed underhigh vacuum for 5 hours for complete removal of the solvent. The driedlipid film is hydrated with 0.32 mol/L mannitol. This is followed bysonication using a sonoporation unit with 1-Mhz frequency for 5 min in awater bath. The mixture is then transferred to a new glass vial andcapped with an open screw cap containing Teflon-covered silicon rubbersepta. The desired gas (e.g., octafluorocyclobutane) is then introducedinto this vial by injection using a syringe through its cap. The gas andliposome dispersion is then pressurized to a supra-atmospheric levelranging from 3 to 9 atms depending on the composition of the desiredlipid dispersion. This pressurized system is incubated for 30 minutes atroom temperature and then frozen by cooling to −78° C. in dry ice for atleast another 30 minutes. After this incubation, the pressure isreleased by unscrewing the cap immediately upon its removal from dryice. The depressurized frozen liposomes are then thawed by exposure toroom air. The final product will be ready for use when its temperaturereaches at least 24° C. (usually after 10 minutes). Additional compoundssuch as free fatty acids or other protein ligands can be loaded atvarious points along this procedure (e.g., mixing with mannitol at thehydrating step).

Due to the versatility of these phospholipid microbubbles and thewide-ranging health effects of omega-3 PUFA, the present invention hasmultiple potential therapeutic applications. In an aspect of the presentinvention, phospholipid microbubbles are custom-formulated for use inthe treatment of acute myocardial infarction and the prevention of itsarrhythmic complications. For this application, phospholipidmicrobubbles having a phospholipid shell comprising a mixture of PS andPC head groups linked to a non-polar fatty acid tail mixture of omega-3PUFA including EPA and DHA are specifically fashioned. Thesephospholipid microbubbles containing a mixture of PS-DHA, PS-EPA,PC-DHA, PC-EPA can be given to a patient before, during and/or after amyocardial infarction. The beneficial effects of omega-3 PUFA in thislife-threatening condition are myriad and include the reduction ofinfarct size, prevention of atrial and ventricular arrhythmias, decreasein reperfusion injury, promotion of the healing process, prevention ofsudden death, and ultimately translating to the prolongation ofsurvival. (Jacobson T A. Secondary Prevention of Coronary Artery Diseasewith Omega-3 Fatty Acids. The American Journal of Cardiology 2006;98:61-70, and Jung U J, Torrejon C, Tighe A P, Deckelbaum R J. n-3 Fattyacids and cardiovascular disease: mechanisms underlying beneficialeffects. Am J Clin Nutr 2008; 87:2003S-9S). In addition, omega-3 PUFAcan exert anti-atherosclerotic, anti-inflammatory and anti-thromboticeffects on the ruptured culprit coronary plaque that precipitated themyocardial infarction. (Robinson J G, Stone N J. Antiatherosclerotic andantithrombotic effects of omega-3 fatty acids. Am J Cardiol 2006;98:39i-49i). Therefore, these phospholipid microbubbles can be used tostabilize inflamed or eroded atherosclerotic plaque during an acutecoronary event or during a transient cerebral ischemia or infarction.(Thies F, Garry J M C, Yaqoob P, et al. Association of n-3polyunsaturated fatty acids with stability of atherosclerotic plaques: arandomised controlled trial. The Lancet 2003; 361:477-85).

In Vivo Experiments

One of the aforementioned effects associated with the present inventionwas tested in a canine model of myocardial infarction. Specifically,phospholipid microbubbles containing omega-3 PUFA were custom-formulatedto test for their purported anti-arrhythmic effects in this establishedpost-infarct animal model. The omega-3 PUFA-containing phospholipidsused in this formulation were derived from a natural marine sourcecalled Antarctic krill (Euphausia superba). The omega-3 PUFA-containingphospholipids extracted from Antarctic krill comprise predominantly ofPC and PS forms of DHA and EPA. For microbubbles preparation, 30milligram of these omega-3 PUFA-containing phospholipids was mixed with1 mg of poly(ethyleneglycol) stearate (Sigma Chemical Co) and dissolvedin normal saline (0.9%) to a final volume of 1 milliliter. Thisdispersion was then transferred to a glass vial and sonicated under roomair for 40 seconds. The final concentration of microbubbles generated bythis method was estimated to be 10⁷/ml. One milliliter of this freshlyprepared microbubbles solution was used to test for its ability toterminate ventricular tachycardia, a life-threatening arrhythmia thatfrequently develops after a myocardial infarction.

To induce ventricular tachycardia, a myocardial infarction was producedin a mongrel dog by ligation of the proximal left anterior descendingcoronary artery under anesthesia with pentobarbital sodium (20 to 25mg/kg IV). The successful creation of myocardial infarction wasascertained by direct visualization of an anterior wall motionabnormality by echocardiography. The cardiac rhythm was continuouslymonitored and recorded via a five-lead surface electrocardiogram using atelemetry unit throughout the experiment. Ventricular tachycardiadeveloped at 15 minutes into the experiment, upon which a 1-ml bolus ofmicrobubbles containing omega-3 PUFA was immediately injected via anintravenous femoral catheter together with simultaneous application ofultrasound over the region of infarction. Within a few seconds aftermicrobubbles injection, the abnormal ventricular tachycardia revertedback to normal sinus rhythm. This dramatic pharmacologic conversion wasrecorded and is shown in FIG. 2. After microbubbles administration, thedog remained in sinus rhythm without further arrhythmias for anotherhour.

In another experiment, an experimental dog developed an anterior wallmyocardial infarction after ligation of the left anterior descendingcoronary artery. About 30 minutes after the infarction, the dogdeveloped ventricular fibrillation. A 0.5 ml bolus of microbubblescontaining omega-3 PUFA was administered intravenously which immediatelyrestored the abnormal rhythm back to normal sinus rhythm for 1 beat.Although transient, this unusual event provided evidence of a positiveanti-arrhythmic effect associated with the administration of thePUFA-containing microbubbles in the dog. However, the dog's rhythm thendegenerated back into ventricular fibrillation. Because of hemodynamicinstability, electrical defibrillation was performed twice in rapidsequence without effect. Another 0.5 ml bolus of PUFA-containingmicrobubbles was injected and electrical defibrillation was subsequentlyattempted, which then successfully restored sinus rhythm back to normal.This course of events suggests that PUFA-containing microbubbles can beuseful in lowering the electrical threshold needed for successfuldefibrillation.

In another experiment, an experimental dog developed ventricular flutterand fibrillation after an extensive myocardial infarction involving alarge territory of the anterior wall as visualized by echocardiographyafter coronary ligation. Intravenous injection of a 1 ml bolus ofmicrobubbles containing omega-3 PUFA failed to restore sinus rhythm.Subsequent electrical defibrillation in 3 consecutive attempts was alsounsuccessful. As a last measure, direct manual massage of the exposedheart also failed to restore the rhythm. Finally, another 1 ml bolus wasinjected after which manual massage successfully restored the rhythmback to normal. Because of the extensive nature of the infarction, thedog suffered from severe circulatory failure which prevented effectiveintravenous delivery of the PUFA-containing microbubbles. In this severecirculatory failure scenario, a larger dose combined with direct manualmassage made it possible for the PUFA-containing microbubbles to exertits anti-arrhythmic effect.

Although specific embodiments have been illustrated and describedherein, it will be appreciated by those of ordinary skill in the artthat a variety of alternate and/or equivalent implementations may besubstituted for the specific embodiments shown and described withoutdeparting from the scope of the present invention. This application isintended to cover any adaptations or variations of the specificembodiments discussed herein. Therefore, it is intended that thisinvention be limited only by the claims and the equivalents thereof.

What is claimed is:
 1. A phospholipid microbubble comprising a shellwhich comprises a plurality of polyunsaturated fatty acid(“PUFA”)-containing phospholipids, and a core of paramagnetic gassurrounded by said shell comprising said plurality of PUFA-containingphospholipids.
 2. The phospholipid microbubble of claim 1, wherein saidplurality of PUFA-containing phospholipids comprising a plurality ofpolar head groups, said plurality of polar head groups comprisingphosphatidylserine (“PS”), phosphadtidylcholine (“PC”),phosphatidyl-ethanolamine (“PE”), phosphatidylinositol (“PI”), or acombination thereof.
 3. The phospholipid microbubble of claim 1, whereinsaid plurality of PUFA-containing phospholipids comprising a pluralityof non-polar fatty acid tails, said plurality of non-polar fatty acidtails comprising an omega-3 PUFA, an omega-3 PUFA precursor, an omega-3PUFA-derived metabolite, an omega-6 PUFA, an omega-9 PUFA, a conjugatedlinoleic acid, a conjugated linoleic acid isomer, or a combinationthereof.
 4. The phospholipid microbubble of claim 3, wherein saidomega-3 PUFA is selected from eicosapentaenoic acid (“EPA”),docosahexaenoic acid (“DHA”), or a combination thereof.
 5. Thephospholipid microbubble of claim 3, wherein said omega-3 PUFA precursoris an □-linolenic acid.
 6. The phospholipid microbubble of claim 3,wherein said omega-3 PUFA-derived metabolite is selected from aresolvin, a neuroprotectin, a lipoxin, a neuroprostane, or a combinationthereof.
 7. The phospholipid microbubble of claim 1, wherein saidparamagnetic gas is selected from a perfluorocarbon compound, xenon, orhyperpolarized xenon.
 8. The phospholipid microbubble of claim 1,wherein said phospholipid microbubble in a micellar form or a liposomalform.
 9. The phospholipid microbubble of claim 1, wherein said pluralityof PUFA-containing phospholipids comprises at least one PS-DHAphospholipid, PS-EPA phospholipid, PC-DHA phospholipid, or PC-EPAphospholipid.
 10. The phospholipid microbubble of claim 1, wherein saidphospholipid microbubble further comprises a drug, a fat-solublecompound, an antioxidant, an antibody or a fragment thereof, or aspecific ligand is conjugated to said phospholipid microbubble.
 11. Amethod of delivering a prophylactically or therapeutically effectiveamount of PUFA to an area of disease or injury in a subject, comprisingthe steps of: administering to said subject a plurality of phospholipidmicrobubbles which comprises a shell comprising a plurality ofPUFA-containing phospholipids, and a core of paramagnetic gas surroundedby said shell comprising said plurality of PUFA-containingphospholipids; allowing said phospholipid microbubbles to reach saidarea of disease or injury; and applying ultrasound to said area ofdisease or injury to explode said phospholipid microbubbles.
 12. Themethod of claim 11, wherein said area of disease or injury is the heart.13. The method of claim 11, wherein said plurality of phospholipidmicrobubbles comprises a micellar form of said phospholipidmicrobubbles, a liposomal form of phospholipid microbubbles, or acombination thereof.
 14. A method of preventing or treating a disease ina subject, comprising the steps of: administering to said subject aprophylatically or therapeutic effective amount of a plurality ofphospholipid microbubbles which comprises a shell comprising a pluralityof PUFA-containing phospholipids, and a core of paramagnetic gassurrounded by said shell comprising said plurality of PUFA-containingphospholipids; and applying ultrasound to said phospholipid microbubblesto explode said phospholipid microbubbles in said subject.
 15. Themethod of claim 14, wherein said disease is lupus erythematosis,multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerativecolitis, psoriasis, diabetes mellitus, prostate cancer, breast cancer,depression, Alzheimer's disease or myocardial infarction.
 16. The methodof claim 14, wherein said administration is a single intravenousadministration of said plurality of phospholipid microbubbles.
 17. Themethod of claim 14, wherein said plurality of PUFA-containingphospholipids comprising at least one PS-DHA phospholipid, PS-EPAphospholipid, PC-DHA phospholipid, or PC-EPA phospholipid.
 18. Themethod of claim 14, wherein said plurality of phospholipid microbubblescomprises a micellar form of said phospholipid microbubbles, a liposomalform of said phospholipid microbubbles, or a combination thereof. 19.The method of claim 14, wherein said administration is aco-administration of a micellar form of said phospholipid microbubblesand a liposomal form of said phospholipid microbubbles.
 20. The methodof claim 19, wherein said co-administration is simultaneous, concurrentor sequential administration of said micellar form of said phospholipidmicrobubbles and said liposomal form of said phospholipid microbubbles.